Cell culture studies of neuronal outgrowth and growth cone guidance are facilitated by the ability to engineer micropatterned substrates (Fedoroff, S. and Richardson, A., Protocols for Neur. Cell Cult., pp. 384, 2001). One approach is soft lithography using an elastomeric material such as polydimethylsiloxane (PDMS) to cast stamps on silicon wafers that are patterned by conventional lithographic methods. The PDMS stamp is coated with a solution of molecules and then pressed directly onto surfaces to be patterned. Patterns can be made using many different materials, for example, proteins and alkanethiols.
An uncoated PDMS stamp can also be placed directly on a surface to pattern the binding of a solution of molecules. The stamp may then be removed to allow binding of a second solution of molecules in the remaining bare areas. This approach was first developed by Bonhoeffer (Vielmetter, J., et al., Exp. Brain. Res. 81:283-7, 1990) and used to study growth cone dynamics at borders between laminin and fibronectin (Gomez, T. M. and Letourneau, P. C., J. Neurosci. 14:5959-72, 1994). A similar method was developed for studies of myosin II activity in growth cones at borders between laminin and poly-ornithine (Turney, S. G. and Bridgman, P. C., Nat. Neurosci. 8:717-9, 2005).
Although soft lithography methods are promising, a number of problems remain to be solved. Registration is an issue for multilayer fabrication due to distortion that is intrinsic to elastomeric materials. To limit defects arising from dust particles fabrication must be performed in a clean room environment. Another issue is maximizing the transfer of molecules from the stamp to the surface. Finally, patterns cannot be altered dynamically due to the time it takes to create or modify a stamp.
Maskless fabrication methods offer the possibility of modifying patterns faster and at less cost than methods based on conventional photolithography. One such method is to “print” proteins on a coated glass surface using inkjet printer technology. Proteins such as extracellular matrix (ECM) components, antibodies, enzymes or receptors are deposited in picoliter droplets, requiring less protein than is used in traditional microtitre dish assays. However, the proteins often lose their biological activity as a result of being dried on the glass surface.
Investigations into the mechanisms underlying cellular behavior such as growth and differentiation are enhanced by the ability to control the environment at a microscopic level. Molecules such as proteins that are known to affect cellular behavior can be laid down in patterns on glass or plastic surfaces. However, the patterns are often fixed and are limited in terms of the detail, complexity and spatial resolution that can be achieved. Photolithography methods are somewhat more flexible and have been used to create patterns of macromolecules (Herbert, C. B., et al., Chem. Biol. 4:731-7, 1997; Kleinfeld, D., et al., J. Neurosci. 8:4098-120, 1988). Nevertheless these methods are time consuming, and a need remains for improved methods to create complex micropatterns and micropatterns that can be modified rapidly in response to changing experimental demands. In particular, methods are needed for generating micropatterns of macromolecules on a substrate in the presence of living cells.
Illumination of cells with laser light through a microscope is facilitated with the use of appropriate culture dishes and lids. Some previous designs of culture dish lids include lids which, while reducing condensation, require laser light to pass through plastic, which can distort the optics or absorb the laser light in an undesirable manner (Turney, S. G. and Bridgman, P. C. 2005. Nature Neurosci. 8:717-719).